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The Journal of Biological Chemistry Dec 1988Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic...
Three glucuronic acid-rich dermatan sulfate proteoglycans (DS-PGs) have been isolated by chromatographic and electrophoretic techniques from cultures of bovine aortic endothelial cells and characterized structurally. The smallest of the DS-PGs (DS-II) has an apparent Mr of approximately 100,000 and glycosaminoglycan chains of Mr approximately 29,000. Core glycoprotein samples prepared by chondroitin ABC lyase digestion run as doublets of Mr = 45,000 and 48,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A decrease in core size is apparent after N-glycanase digestion, or when DS-PG is isolated from tunicamycin-treated cultures, providing evidence that the core protein is N-glycosylated. Isolated DS-II shows evidence of self-association when subjected to liquid chromatography under conditions of reduced ionic strength, but not during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, DS-II, but not other endothelial cell DS-PG subclasses, is bound by an antibody against human skin fibroblast DS-PG, indicating that this DS-PG belongs to a family of widely distributed small DS-PGs, previously isolated from various connective tissues. A slightly larger (Mr approximately 220,000) DS-PG (DS-I) can be separated from DS-II by preparative electrophoresis. Despite similarities in core size and extent of N-glycosylation between DS-I and DS-II, DS-I shows only limited ability to self-associate, and does not interact with the anti-fibroblast DS-PG antibody. DS-I glycosaminoglycan chains are also smaller (Mr approximately 18,000) than those from DS-II, similar in size to the chains borne by the DS-PG subclass of largest size (high molecular weight (HMW)-DS). HMW-DS, which predominated in cell layer extracts, runs with a Kav of 0.45 on Sepharose CL-2B and is estimated to have an Mr greater than 700,000. Reduction and alkylation of HMW-DS indicates that it forms disulfide-bonded aggregates with other matrical proteins within the cell layer. HMW-DS displayed multiple protein cores (Mr greater than 200,000) upon chondroitin ABC lyase treatment. Despite some similarity in size to the family of large, aggregating chondroitin sulfate proteoglycans and DS-PGs, immunological evidence suggests that it lacks a hyaluronic acid binding region.
Topics: Animals; Cattle; Cells, Cultured; Chondroitin; Chondroitin Sulfate Proteoglycans; Chromatography, Gel; Dermatan Sulfate; Endothelium, Vascular; Fluorometry; Immunohistochemistry; Methionine; Proteoglycans
PubMed: 3198623
DOI: No ID Found -
The Journal of Biological Chemistry Oct 2001The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and... (Comparative Study)
Comparative Study
The content and fine structure of keratan and chondroitin/dermatan sulfate in normal human corneas and corneas affected by macular corneal dystrophies (MCD) types I and II were examined by fluorophore-assisted carbohydrate electrophoresis. Normal tissues (n = 11) contained 15 microg of keratan sulfate and 8 microg of chondroitin/dermatan sulfate per mg dry weight. Keratan sulfates consisted of approximately 4% unsulfated, 42% monosulfated, and 54% disulfated disaccharides with number of average chain lengths of approximately 14 disaccharides. Chondroitin/dermatan sulfates were significantly longer, approximately 40 disaccharides per chain, and consisted of approximately 64% unsulfated, 28% 4-sulfated, and 8% 6-sulfated disaccharides. The fine structural parameters were altered in all diseased tissues. Keratan sulfate chain size was reduced to 3-4 disaccharides; chain sulfation was absent in MCD type I corneas and cartilages, and sulfation of both GlcNAc and Gal was significantly reduced in MCD type II. Chondroitin/dermatan sulfate chain sizes were also decreased in all diseased corneas to approximately 15 disaccharides, and the contents of 4- and 6-sulfated disaccharides were proportionally increased. Tissue concentrations (nanomole of chains per mg dry weight) of all glycosaminoglycan types were affected in the disease types. Keratan sulfate chain concentrations were reduced by approximately 24 and approximately 75% in type I corneas and cartilages, respectively, and by approximately 50% in type II corneas. Conversely, chondroitin/dermatan sulfate chain concentrations were increased by 60-70% in types I and II corneas. Such changes imply a modified tissue content of individual proteoglycans and/or an altered efficiency of chain substitution on the core proteins. Together with the finding that hyaluronan, not normally present in healthy adult corneas, was also detected in both disease subtypes, the data support the conclusion that a wide range of keratocyte-specific proteoglycan and glycosaminoglycan remodeling processes are activated during degeneration of the stromal matrix in the macular corneal dystrophies.
Topics: Acetylglucosamine; Adult; Aged; Carbohydrate Sequence; Cartilage; Chondroitin Sulfates; Cornea; Corneal Dystrophies, Hereditary; Dermatan Sulfate; Fucose; Glycopeptides; Glycosaminoglycans; Humans; Keratan Sulfate; Middle Aged; Molecular Sequence Data; Oligosaccharides
PubMed: 11514545
DOI: 10.1074/jbc.M103227200 -
The Journal of Biological Chemistry Apr 2009A second dermatan sulfate epimerase (DS-epi2) was identified as a homolog of the first epimerase (DS-epi1), which was previously described by our group. DS-epi2 is 1,222...
A second dermatan sulfate epimerase (DS-epi2) was identified as a homolog of the first epimerase (DS-epi1), which was previously described by our group. DS-epi2 is 1,222 amino acids long and has an approximately 700-amino acid N-terminal epimerase domain that is highly conserved between the two enzymes. In addition, the C-terminal portion is predicted to be an O-sulfotransferase domain. In this study we found that DS-epi2 has epimerase activity, which involves conversion of d-glucuronic acid to l-iduronic acid (EC 5.1.3.19), but no O-sulfotransferase activity was detected. In dermatan sulfate, iduronic acid residues are either clustered together in blocks or alternating with glucuronic acid, forming hybrid structures. By using a short interfering RNA approach, we found that DS-epi2 and DS-epi1 are both involved in the biosynthesis of the iduronic acid blocks in fibroblasts and that DS-epi2 can also synthesize the hybrid structures. Both iduronic acid-containing domains have been shown to bind to several growth factors, many of which have biological roles in brain development. DS-epi2 has been genetically linked to bipolar disorder, which suggests that the dermatan sulfate domains generated by a defective enzyme may be involved in the etiology of the disease.
Topics: Amino Acid Sequence; Cell Line; Cells, Cultured; Dermatan Sulfate; Fibroblasts; Glucuronic Acid; Golgi Apparatus; Humans; Iduronic Acid; Molecular Sequence Data; Plasmids; Protein Structure, Tertiary; RNA, Small Interfering; Racemases and Epimerases; Sulfotransferases
PubMed: 19188366
DOI: 10.1074/jbc.M809339200 -
The Journal of Biological Chemistry Jun 1987A proteoglycan was isolated from the human placenta by procedures including affinity chromatography with fibronectin immobilized on agarose. The glycosaminoglycan chains...
A proteoglycan was isolated from the human placenta by procedures including affinity chromatography with fibronectin immobilized on agarose. The glycosaminoglycan chains were found to be composed of heparan sulfate (86%) and dermatan sulfate (14%). The average molecular weights were estimated to be 1.8 X 10(5) for heparan sulfate and 1.2 X 10(5) for dermatan sulfate. Mouse monoclonal antibodies HS42 and HS47 were prepared against the proteoglycan, and examination of the specificity of these antibodies indicated that they recognized the core protein portion. The binding specificity, as studied by the solid phase enzyme-linked immunoassay with monoclonal antibody HS47, indicated that the proteoglycan bound to solid phase fibronectin and to laminin, but not to collagen types I, II, and IV or gelatin. Competitive immunoassays suggested that the proteoglycan bound weakly to the liquid phase-soluble fibronectin. These studies also indicated that the core protein was involved in the interaction between the proteoglycan and solid phase fibronectin. The ubiquitous distribution of this proteoglycan in the human tissues was demonstrated by the immunohistochemical method and thus suggested its important role in the tissue organization and function.
Topics: Amino Acids; Antibodies, Monoclonal; Chondroitin; Chromatography, Affinity; Dermatan Sulfate; Fibronectins; Fluorescent Antibody Technique; Glycosaminoglycans; Heparitin Sulfate; Immunoenzyme Techniques; Placenta; Proteoglycans; Receptors, Fibronectin; Receptors, Immunologic
PubMed: 2954956
DOI: No ID Found -
Journal of Structural Biology Apr 2007The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been...
The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been suggested to contribute to the mechanical stability and material properties of connective tissues. The spatial distribution and orientation of DS within the tissue are relevant to these mechanical roles, but measurements of length and orientation from 2D transmission electron microscopy (TEM) are prone to errors from projection. The objectives of this study were to construct a 3D geometric model of DS GAGs and collagen fibrils, and to use the model to interpret TEM measurements of the spatial orientation and length of DS GAGs in the medial collateral ligament of the human knee. DS was distinguished from other sulfated GAGs by treating tissue with chondroitinase B, an enzyme that selectively degrades DS. An image processing pipeline was developed to analyze the TEM micrographs. The 3D model of collagen and GAGs quantified the projection error in the 2D TEM measurements. Model predictions of 3D GAG orientation were highly sensitive to the assumed GAG length distribution, with the baseline input distribution of 69+/-23 nm providing the best predictions of the angle measurements from TEM micrographs. The corresponding orientation distribution for DS GAGs was maximal at orientations orthogonal to the collagen fibrils, tapering to near zero with axial alignment. Sulfated GAGs that remained after chondroitinase B treatment were preferentially aligned along the collagen fibril. DS therefore appears more likely to bridge the interfibrillar gap than non-DS GAGs. In addition to providing quantitative data for DS GAG length and orientation in the human MCL, this study demonstrates how a 3D geometric model can be used to provide a priori information for interpretation of geometric measurements from 2D micrographs.
Topics: Chondroitinases and Chondroitin Lyases; Collateral Ligaments; Dermatan Sulfate; Glycosaminoglycans; Humans; Imaging, Three-Dimensional; Microscopy, Electron, Transmission
PubMed: 17150374
DOI: 10.1016/j.jsb.2006.10.008 -
Journal of Biochemistry Feb 1975A simple chemical method for the determination of individual mucopolysaccharides in mixtures of dermatan sulfate and chondroitin sulfates by means of a single reagent...
A simple chemical method for the determination of individual mucopolysaccharides in mixtures of dermatan sulfate and chondroitin sulfates by means of a single reagent was established, utilizing the difference in reaction rates of these polysaccharides with orcinol. To each 1 ml of a sample mixture of standard dermatan sulfate and standard chondroitin sulfate (either 4- or 6-sulfate) was added 3 ml of orcinol reagent and the resulting solution was heated in a boiling-water bath. After 20 and 60 min reaction, absorbances at 660 nm were measured and the concentrations of individual mucopolysaccharides were calculated. High reproducibility was observed for the determination of dermatan sulfate in the presence of chondroitin sulfates. In addition, orcinol reaction for 90 min employing D-glucuronolactone as a standard appeared to be of practical value in the estimation of the uronic acid content of these mucopolysaccharides.
Topics: Carbazoles; Chondroitin; Dermatan Sulfate; Hydrolysis; Indicators and Reagents; Kinetics; Methods; Spectrophotometry; Time Factors; Uronic Acids
PubMed: 1126924
DOI: 10.1093/oxfordjournals.jbchem.a130738 -
Molecules (Basel, Switzerland) Sep 2022Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western...
Monkeypox virus (MPXV), a member of the Orthopoxvirus genus, has begun to spread into many countries worldwide. While the prevalence of monkeypox in Central and Western Africa is well-known, the recent rise in the number of cases spread through intimate personal contact, particularly in the United States, poses a grave international threat. Previous studies have shown that cell-surface heparan sulfate (HS) is important for vaccinia virus (VACV) infection, particularly the binding of VACV A27, which appears to mediate the binding of virus to cellular HS. Some other glycosaminoglycans (GAGs) also bind to proteins on Orthopoxviruses. In this study, by using surface plasmon resonance, we demonstrated that MPXV A29 protein (a homolog of VACV A27) binds to GAGs including heparin and chondroitin sulfate/dermatan sulfate. The negative charges on GAGs are important for GAG-MPXV A29 interaction. GAG analogs, pentosan polysulfate and mucopolysaccharide polysulfate, show strong inhibition of MPXV A29-heparin interaction. A detailed understanding on the molecular interactions involved in this disease should accelerate the development of therapeutics and drugs for the treatment of MPXV.
Topics: Chondroitin Sulfates; Dermatan Sulfate; Glycosaminoglycans; Heparin; Heparitin Sulfate; Monkeypox virus; Pentosan Sulfuric Polyester; Surface Plasmon Resonance; Vaccinia virus
PubMed: 36144634
DOI: 10.3390/molecules27185898 -
The Journal of Biological Chemistry Dec 1986Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules...
Rat glomerular heparan sulfate (HS) and dermatan sulfate (DS) proteoglycan synthesis was studied in vitro and in vivo. Incorporation of [35S]sulfate into macromolecules was linear over 16 h in vitro, and DS was the predominant glycosaminoglycan (GAG), while HS dominated in vivo incubations. Proteoglycans were found in the bottom 2/5 (high density) CsCl gradient fractions and eluted as two overlapping peaks from DEAE-Sephacel columns. The proportion of low density 35S-glycoproteins and 35S-proteoglycans increased with time. Two high buoyant density HS proteoglycans were extracted from glomeruli and eluted in DEAE peak I. The first, HS-tIA, had an Mr of 130 X 10(3) with Mr 12.5 X 10(3) GAG chains. This proteoglycan was released from the tissue by trypsin and was partially displaced by heparin treatment. In addition, it was rapidly released into the medium of label-chase experiments after which it migrated slightly more rapidly than HS-tIA in gels, with HS chains similar in length to its tissue counterpart. The second, HS-tIB, had an Mr of 8.6 X 10(3) with little or no attached protein. This proteoglycan was characterized as intracellular as it resisted release by trypsin treatment or heparin extraction in medium and was not detected in the medium of label-chase experiments. Two tissue DS proteoglycans were characterized. The first, DS-tIA, co-purified with HS-tIA and was the predominant proteoglycan synthesized during 4-h in vitro incubations. Like HS-tIA, it was rapidly released into medium and displaced from cell surfaces or tissue "receptors" by heparin or trypsin treatments. A second, Sepharose CL-6B-excluded DS proteoglycan from DEAE peak II, DS-tII, accumulated in tissue over 16 h in vitro. This proteoglycan was self-associating and contained clusters of iduronic acid residues along its Mr 26 X 10(3) DS chains. It resisted extraction from the tissue with heparin, trypsin, and detergent. No DS-tII was detected in the incubation medium. Instead, medium proteoglycans eluted as single Sepharose CL-6B-included peaks. DS chains from medium proteoglycans were shorter (Mr 18 X 10(3)) and had more regularly spaced iduronic acid residues than GAGs from DS-tII. The length and sulfation patterns of DS-mII GAG were similar to GAG from DS-tIA. Thus, glomeruli rapidly synthesized and released Sepharose CL-6B-included heparin-displaceable DS and HS proteoglycans while retaining a Sepharose CL-6B-excluded self-associating DS proteoglycan and an intracellular HS.
Topics: Animals; Chondroitin; Chondroitin Sulfate Proteoglycans; Chromatography, Ion Exchange; Dermatan Sulfate; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; In Vitro Techniques; Kidney Glomerulus; Kinetics; Male; Proteoglycans; Rats; Rats, Inbred Strains; Sulfates; Sulfur Radioisotopes
PubMed: 2946688
DOI: No ID Found -
PloS One 2015Shark fin, used as a food, is a rich source of glycosaminoglyans (GAGs), acidic polysaccharides having important biological activities, suggesting their nutraceutical...
Composition of glycosaminoglycans in elasmobranchs including several deep-sea sharks: identification of chondroitin/dermatan sulfate from the dried fins of Isurus oxyrinchus and Prionace glauca.
Shark fin, used as a food, is a rich source of glycosaminoglyans (GAGs), acidic polysaccharides having important biological activities, suggesting their nutraceutical and pharmaceutical application. A comprehensive survey of GAGs derived from the fin was performed on 11 elasmobranchs, including several deep sea sharks. Chondroitin sulfate (CS) and hyaluronic acid (HA) were found in Isurus oxyrinchus, Prionace glauca, Scyliorhinus torazame, Deania calcea, Chlamydoselachus anguineus, Mitsukurina owatoni, Mustelus griseus and Dasyatis akajei, respectively. CS was only found from Chimaera phantasma, Dalatias licha, and Odontaspis ferox, respectively. Characteristic disaccharide units of most of the CS were comprised of C- and D-type units. Interestingly, substantial amount of CS/dermatan sulfate (DS) was found in the dried fin (without skin and cartilage) of Isurus oxyrinchus and Prionace glauca. 1H-NMR analysis showed that the composition of glucuronic acid (GlcA) and iduronic acid (IdoA) in shark CS/DS was 41.2% and 58.8% (Isurus oxyrinchus), 36.1% and 63.9% (Prionace glauca), respectively. Furthermore, a substantial proportion of this CS/DS consisted of E-, B- and D-type units. Shark CS/DS stimulated neurite outgrowth of hippocampal neurons at a similar level as DS derived from invertebrate species. Midkine and pleiotrophin interact strongly with CS/DS from Isurus oxyrinchus and Prionace glauca, affording Kd values of 1.07 nM, 6.25 nM and 1.70 nM, 1.88 nM, respectively. These results strongly suggest that the IdoA-rich domain of CS/DS is required for neurite outgrowth activity. A detailed examination of oligosaccharide residues, produced by chondroitinase ACII digestion, suggested that the IdoA and B-type units as well as A- and C-type units were found in clusters in shark CS/DS. In addition, it was discovered that the contents of B-type units in these IdoA-rich domain increased in a length dependent manner, while C- and D-type units were located particularly in the immediate vicinity of the IdoA-rich domain.
Topics: Animal Fins; Animals; Chondroitin Sulfates; Dermatan Sulfate; Mice; Neurites; Sharks
PubMed: 25803296
DOI: 10.1371/journal.pone.0120860 -
Molecular and Cellular Biology Oct 2009Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the...
Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.
Topics: Animals; Carbohydrate Epimerases; Collagen; Decorin; Dermatan Sulfate; Extracellular Matrix Proteins; Iduronic Acid; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Proteoglycans; Skin
PubMed: 19687302
DOI: 10.1128/MCB.00430-09